An extrachromosomal DNA molecule of unknown function, designated multicopy single-stranded DNA or msDNA, is of keen interest for its unusual structural features. Occurring at about 700 copies per genome, msDNA was first discovered in the soil bacterium Myxococcus xanthus (Yee et al., 1984). M. xanthus, a myxobacterium, is a Gram-negative rod which, upon starvation, aggregates into a multicellular state followed by a complex morphogensis into a fruiting body containing spores ( Kaiser, 1986; Shimkets, 1987). In addition to several other diverse species of myxobacteria which also contain msDNA (Dhundale et al., 1985), msDNA has recently been found in a clinical isolate of Escherichia coli (Lampson et al., 1989).
Nucleotide sequencing of msDNA from M. xanthus (msDNA-Mx162) revealed that the molecule is composed of a single-strand of DNA of 162 bases, and attached to the 5' end is a unique, branched RNA molecule of 77 nucleotides. The single-stranded RNA (msRNA) is linked to the 5' end of the DNA strand at the 20th granosine residue via a unique 2', 5' phosphodiester bond (Dhundale et at., 1987).
A nearly identical molecule has been characterized from another myxobacterium Stigmatella aurantiaca (Furuichi et al., 1987a,b). In addition, a smaller species of msDNA (designated msDNA-Mx65), which coexists in M. xanthus, also shares the same key structural features with the larger msDNA including a branched rG residue, a DNA-RNA hybrid at the 3' ends of the msDNA and msdRNA, and stem-and-loop structures in RNA and DNA strands. However, it is encoded by a separate gene and is composed of a different nucleotide sequence (Dhundale et al., 1988b). A single copy chromosomal locus contains a gene, msd, which encodes the DNA strand of msDNA-Mx162 and a gene, msr, which encodes the RNA molecule. The two genes are convergently situated (5' to 3') such that their respective 3' ends overlap by 8 bases. In a clinical strain of E. coli, msDNA (msDNA-Ec67) which shares the same secondary structures but has a different primary sequence from the myxobacterial msDNAs, is also encoded by a single chromosomal locus. Situated about 50 bases from this locus is an open reading frame (ORF) which displays sequences comparable to known retroviral reverse transcriptases (RT) (Lampson et al., 1989). In Inouye et al., 1989, an ORF located 77 bases upstream of the msd gene of M. xanthus is described. Like that of E. coli, this ORF also shows significant amino acid sequence similarity to retroviral RT.
From S1 mapping experiments, the msdRNA of M. xanthus encoded by the msr gene is derived from a much longer precursor transcript which is likely to form a stable stem-and-loop structure (Dhundale et al., 1987). In addition, we proposed a model to explain the synthesis of the msDNA molecule in which the folded precursor RNA serves as both a primer and template for the synthesis of the branch-linked DNA strand via a reverse transcriptase activity. This application describes a cell-free system utilizing permeablized cells treated with phenethyl alcohol to study the biosynthesis of msDNA in M. xanthus. In this system [.alpha.-.sup.= p]dCPT is incorporated into msDNA. Intermediate structures were identified by interrupting the synthesis of msDNA with dideoxyribonucleotides. Analysis of the intermediate species provides compelling evidence that msDNA is synthesized from an RNA template by RT activity, and that there is a very precise coupling between RT activity and ribonuclease H (RNase H) activity.